1. Field of the Invention
The present invention relates to the field of biosensors for detecting molecular interactions and, further, to devices having nanopores to which are attached active biological molecules.
2. Related Art
As described in US PGPUB 2005/0186629, manipulating matter at the nanometer scale is important for many electronic, chemical and biological advances (See Li et al., “Ion beam sculpting at nanometer length scales,” Nature, 2001, 412: 166-169). Such techniques as “ion beam sculpting” have shown promise in fabricating molecule scale holes and nanopores in thin insulating membranes. These pores have also been effective in localizing molecular-scale electrical junctions and switches.
Artificial nanopores have been fabricated by a variety of research groups with a number of materials. Generally, the approach is to fabricate these nanopores in a solid-state material or a thin freestanding diaphragm of material supported on a frame of thick silicon to form a nanopore chip. Some materials that have been used to date for the diaphragm material include silicon nitride and silicon dioxide.
The flow of materials through nanopores can also be externally regulated. What is believed to be the first artificial voltage-gated molecular nanosieve was fabricated (Matsuhiko Nishizawa, Vinod P. Menon, Charles R. Martin, “Metal nanotubule membranes with electrochemically switchable ion-transport selectivity,” Science, 5 May 1995, 268:700-702) at Colorado State University in 1995. This membrane contained an array of cylindrical gold nanotubules with inside diameters as small as 1.6 nanometers. When the tubules are positively charged, positive ions are excluded and only negative ions are transported through the membrane. When the membrane receives a negative voltage, only positive ions can pass. Similar nanodevices may combine voltage gating with pore size, shape, and charge constraints to achieve precise control of ion transport with significant molecular specificity. A sensitive ion channel switch biosensor was built by an Australian research group (B. Cornell, V. Braach-Maksvytis, L. King, P. Osman, B. Raguse, L. Wieczorek, R. Pace, “A biosensor that uses ion-channel switches,” Nature, 5 Jun. 1997, 387:580-583.) The scientists estimated that their sensor could detect a minute change in chemical concentration equivalent to one part in roughly 1018.
Experiments have been conducted using an electric field to drive a variety of RNA and DNA polymers through the central nanopore of an alpha-hemolysin protein channel mounted in a lipid bilayer similar to the outer membrane of a living cell (A. Meller, L. Nivon, E. Brandin, J. Golovchenko, D. Branton, “Rapid nanopore discrimination between single polynucleotide molecules,” Proc. Natl. Acad. Sci. (USA), 1 Feb. 2000, 97:1079-1084). Researchers have reported that the individual nucleotides comprising the polynucleotide strands must be passing single-file through the 2.6 nanometer-wide nanopore, and that changes in ionic current could be used to measure polymer length, and that the nanopore could be used to rapidly discriminate between pyrimidine and purine segments (the two types of nucleotide bases) along a single RNA molecule. Nanopore devices which can discriminate between purine and pyrimidine molecules have been reported (D. W. Deamer, M. Akeson, “Nanopores and nucleic acids: prospects for ultrarapid sequencing,” Trends Biotechnol, April 2000, 18:147-15). Differences in ionic blockage current were measured. Because nanopores can rapidly discriminate and characterize DNA polymers at low copy number, future refinements of this experimental approach may eventually provide a low-cost high-throughput method for very rapid genome sequencing.
Although nanopore-based sensors have been explored for single nucleic acid molecule sequencing, they still cannot claim success. But they offer one of the most promising approaches for structural analysis of single molecules of few nanometers diameter.